RNA interference (RNAi) is a naturally occurring regulatory mechanism of most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules to direct homology-dependent gene silencing. Its discovery by Fire and Mello in the worm C. elegans {Fire, 1998} was awarded the Nobel prize in 2006. Shortly after its first description, RNAi was also shown to occur in mammalian cells, not through long dsRNAs but by means of double-stranded small interfering RNAs (siRNAs) 21 nucleotides long {Elbashir, 2001}.
The process of RNA interference is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse phyla and flora, where it is called post-transcriptional gene silencing. Since the discovery of RNAi mechanism there has been an explosion of research to uncover new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise “undruggable” with traditional pharmaceutical approaches involving small molecules or proteins.
According to current knowledge, the mechanism of RNAi is initiated when long double stranded RNAs are processed by an RNase III-like protein known as Dicer. The protein Dicer typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains and a double-stranded RNA binding domain (dsRBD) {Collins, 2005} and its activity leads to the processing of the long double stranded RNAs into 21-24 nucleotide double stranded siRNAs with 2 base 3′ overhangs and a 5′ phosphate and 3′ hydroxyl group. The resulting siRNA duplexes are then incorporated into the effector complex known as RNA-induced silencing complex (RISC), where the antisense or guide strand of the siRNA guides RISC to recognize and cleave target mRNA sequences {Elbashir, 2001} upon adenosine-triphosphate (ATP)-dependent unwinding of the double-stranded siRNA molecule through an RNA helicase activity {Nykanen, 2001}. The catalytic activity of RISC, which leads to mRNA degradation, is mediated by the endonuclease Argonaute 2 (AGO2) {Liu, 2004; Song, 2004}. AGO2 belongs to the highly conserved Argonaute family of proteins. Argonaute proteins are ˜100 KDa highly basic proteins that contain two common domains, namely PIWI and PAZ domains {Cerutti, 2000}. The PIWI domain is crucial for the interaction with Dicer and contains the nuclease activity responsible for the cleavage of mRNAs {Song, 2004}. AGO2 uses one strand of the siRNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the guide strand's 5′ end {Elbashir, 2001}. An important step during the activation of RISC is the cleavage of the sense or passenger strand by AGO2, removing this strand from the complex {Rand, 2005}. Crystallography studies analyzing the interaction between the siRNA guide strand and the PIWI domain reveal that it is only nucleotides 2 to 8 that constitute a “seed sequence” that directs target mRNA recognition by RISC, and that a mismatch of a single nucleotide in this sequence may drastically affect silencing capability of the molecule {Ma, 2005; Doench 2004; Lewis, 2003}. Once the mRNA has been cleaved, and due to the presence of unprotected RNA ends in the fragments, the mRNA is further cleaved and degraded by intracellular nucleases and will no longer be translated into proteins {Orban, 2005} while RISC will be recycled for subsequent rounds {Hutvagner, 2002}. This constitutes a catalytic process leading to the selective reduction of specific mRNA molecules and the corresponding proteins. It is possible to exploit this native mechanism for gene silencing with the purpose of regulating any gene(s) of choice by directly delivering siRNA effectors into the cells or tissues, where they will activate RISC and produce a potent and specific silencing of the targeted mRNA.
Many studies have been published describing the ideal features a siRNA should have to achieve maximum effectiveness, regarding length, structure, chemical composition, and sequence. Initial parameters for siRNA design were set out by Tuschl and co-workers in WO02/44321, although many subsequent studies, algorithms and/or improvements have been published since then.
Also, a lot of effort has been put into enhancing siRNA stability as this is perceived as one of the main obstacles for therapy based on siRNA, given the ubiquitous nature of RNAses in biological fluids. One of the main strategies followed for stability enhancement has been the use of modified nucleotides such as 2′-O-methyl nucleotides, 2′-amino nucleotides, nucleotides containing 2′-O or 4′-C methylene bridges. Also, the modification of the ribonucleotide backbone connecting adjacent nucleotides has been described, mainly by the introduction of phosphorothioate modified nucleotides. It seems that enhanced stability is often inversely proportional to efficacy (Parish, 2000), and only a certain number, positions and/or combinations of modified nucleotides may result in a stable silencing compound. As this is an important hurdle within siRNA-based treatments, different studies have been published which describe certain modification patterns showing good results, examples of such include EP1527176, WO2008/050329, WO2008/104978 or WO2009/044392, although many more may be found in the literature.
The Transient Receptor Potential Vanilloid-1 (TRPV1), also called Vanilloid Receptor 1 (VR-1), is a capsaicin-responsive ligand-gated cation channel, that was first discovered in 1997 (Caterina, 1997). TRPV1 is mainly expressed on sensory neurons and serves as a molecular detector for heat, capsaicin, protons, and endovanilloids (Caterina, 2001; Montell, 2002; Baumann, 2000). Although the inventors of the present application have also found TRPV1 expression in tissues from the lacrimal gland and ciliary body.
When TRPV1 is activated by agonists such as capsaicin and other factors such as heat, acidosis, lipoxygenase products or anandamide, calcium enters the cell and pain signals are initiated. Activation of the channel induces neuropeptide release from central and peripheral sensory nerve terminals, resulting in the sensation of pain, neurogenic inflammation, and sometimes, in smooth muscle contraction and cough. As a matter of fact, recent evidence suggests a role of TRPV1 in pain, cough, asthma and urinary incontinence (Jia, 2005). In fact, TRPV1 is a known target for treatments by analgesia in response to pain stimuli. Moreover, treatments designed to reduce expression levels of TRPV1 using different technologies have also been described in WO2004/042046, or (Schubert, 2005), with a focus on the treatment of pain.
Polymodal nociceptors are the most abundant nociceptor type found in the cornea. There exists pharmacological evidence that these receptor fibers express TRPV1 receptor because they respond to capsaicin, heat and acid. Moreover, high doses of capsaicin inactivate the response of corneal polymodal nociceptors to heat and acid whereas mechanical responsiveness remains unaffected. This suggests that TRPV1 receptors present in corneal polymodal nerve endings were selectively inactivated. Therefore, it is likely that an important part of the acute nociceptive response to corneal injury and the sustained pain sensations that accompany inflammatory and irritative processes in this tissue are mediated by TRPV1 activation.
Furthermore, WO2007/045930 describes the use of TRPV1 specific siRNAs for treatment of ocular pathologies related to ocular pain and dry eye syndrome. However, the present invention provides improved products for reducing TRPV1 expression and consequent ocular discomfort. The advantage of treating these conditions with siRNA products vs traditional chemical inhibitors is that treatments based on siRNA will have a longer-lasting effect. This result is due to the fact that once the effector molecule is no longer present, the cell will have to synthesise new receptors from scratch; whereas traditional treatments would leave the levels of receptors on the cell membrane intact.
Due to current life-style, the number of people affected by ocular pathologies related to altered ocular sensitivity is quite high, and is expected to increase with aging of population. Refractive surgery and contact lens use often derive in altered corneal sensitivity and a sensation of dry eye by the patient. This is further aggravated by long working hours looking at computer screens and the use of air-conditioning systems which usually further dry the atmosphere. Also, the quantity and quality of tears decrease with age. Symptoms accompanying dry eye syndromes include itching, burning and irritation of the ocular tissues. A more severe form of dry eye occurs in patients with Sjogren's syndrome. The presence of one or different combinations of these sensations is termed ocular pain within the meaning of the present text. At present dry eye syndrome is estimated to affect over 10 million Americans.